The aim of this study was to analyze the behavioral and physiological reactions of guide dogs in a distressing situation which promotes attachment behaviors towards their blind owners, and to compare such reactions with those of untrained or trainee dogs. The subjects were 57 adult Labrador and Golden retriever dogs (14 males, 43 females) belonging to four different groups: 19 Custody dogs, 13 Apprentice dogs, 10 Guide dogs and 15 Pet dogs. Dogs were tested using the Strange Situation Test, consisting in seven 3-minute episodes in which the dogs were placed in an unfamiliar environment, introduced to an unfamiliar woman and subjected to separation from their human companion. Tests were video-recorded and behaviors were scored using a 5-second point sampling method. Polar Vantage telemetric system was used to record cardiac activity. ANOVAs for repeated measures with groups and breeds as independent variables, showed a more anxious reaction in pet dogs, which revealed a high degree of proximity seeking behavior. Cardiac activity increased during episodes characterized by the exclusive presence of the stranger, but this increase was more conspicuous in guide dogs than in custody and apprentice dogs. Golden retrievers showed more behaviors suggesting distress compared to Labrador retrievers. This study showed that guide dogs, when separated from their blind owner, reveal a controlled behavioral reaction that is however accompanied by a stronger cardiac activation.